An Alkali Tolerant Α-L-Rhamnosidase from Fusarium Moniliforme MTCC

Bioorganic Chemistry 84 (2019) 24–31

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

An alkali tolerant α-L-rhamnosidase from Fusarium moniliforme MTCC-2088 T used in de-rhamnosylation of natural glycosides ⁎ Dhirendra Kumar, Sarita Yadav , Sudha Yadava, K.D.S. Yadav

Department of Chemistry, Deen Dayal Upadhyay Gorakhpur University, Gorakhpur 273009, UP, India

ARTICLE INFO ABSTRACT

Keywords: Analkali tolerant α-L-rhamnosidase has been purified to homogeneity from the culture filtrate of a newfungal Fusarium moniliforme strain, Fusarium moniliforme MTCC-2088, using concentration by ultrafiltration and cation exchange chroma- α-L-Rhamnosidase tography on CM cellulose column. The molecular mass of the purified enzyme has been found to be 36.0 kDa Wine using SDS-PAGE analysis. The Km value using p-nitrophenyl-α-L-rhamnopyranoside as the variable substrate in Prunin 0.2 M sodium phosphate buffer pH10.5 at50 °C was 0.50 mM. The catalytic rate constant was15.6 s−1giving the Isoquercetin values of k /K is 3.12 × 104M−1 s−1. The pH and temperature optima of the enzyme were 10.5 and 50 °C, Hesperetin-glucosidase cat m respectively. The purified enzyme had better stability at 10 °C inbasic pH medium. The enzyme derhamnosylated natural glycosides like naringin to prunin, rutin to isoquercitrin and hesperidin to hesperetin glucoside. The purified α-L-rhamnosidase has potential for enhancement of wine aroma.

1. Introduction L-rhamnosidases from new sources with novel properties and to de- monstrate their applications for different bio transformations. In this α-L-rhamnosidase [E.C.3.1.1.40] selectively derhamnosylates nat- communication, the authors report an alkali resistant α-L-rhamnosidase ural glycosides containing terminalα-L-rhamnose [1,2]. The derham- from Fusarium moniliforme MTCC-2088 which transforms naringin to nosylated products are rare compounds of pharmaceutical importance prunin, hesperidin to hesperetin glucoside and rutin to isoquercitrin [3–11]. α-L-rhamnosidase transforms naringin (4′,5,7trihydroxy flava- and releases L-rhamnose from a wine sample indicating that it has po- none-7-rhamnoglucoside) to prunin (4′5,7trihydroxy flavanone-7-glu- tential for aroma enhancement of wine. coside), which has enhanced bioavailability compared to naringin and its aglycon naringenin (4′5,7,trihydroxy flavanone) while maintaining 2. Materials and methods its bioactivity [3]. The derhamnosylation of hesperidin (hesperetin-7-O- rhamnoglucoside) by α-L-rhamnosidase gives hesperetin-7-O-glucoside, 2.1. Materials which is a rare pharmaceutically important compound [5,6]. α-L- rhamnosidase transforms rutin (quercetin-3-O-rutinoside) to iso- Naringin, rutin, hesperidin, L-rhamnose, p-nitrophenyl-α-L-rhamno- quercitrin (quercetin-3-O-glucoside) which has been reported as a drug pyranoside, and CM cellulose were purchased from Sigma Chemical for a number of diseases due to its non-oxidisable, anti-inflammatory, Company, St. Louis, (USA). The chemicals for gel-electrophoresis in- anti-mutagenetic, anti viral properties and other pharmacological ef- cluding the protein molecular weight markers used in the SDS-PAGE fects [7,8]. Moreover, isoquercitrin is a precursor for the enzymatic and native-PAGE analysis were procured from Bangalore GENEI Pvt. biosynthesis of enzymatically modified isoquercitrin (EMIQ) which has Limited Bangalore (India). All other chemicals were either from Merck been approved as a multiple food additive [12,13]. An alkalitolerant Ltd., Mumbai (India) or from s.d. fine chem. Ltd., Mumbai (India) and thermostable α-L-rhamnosidase from Aspergillus terreus have been re- were used without further purifications.The wine sample was of ported, its recombinant enzyme has been prepared and used for selec- Macleods distillers Ltd. Broxbum (UK) make. tive conversion of rutin to isoquercitrin [14–16]. α-L-rhamnosidases in combination with other glycosidases has been found to improve the 2.2. The fungal strain quality of wine [17,18]. These biotechnological applications of α-L- rhamnosidases have prompted the authors to purify and characterize α- Five fungal strains namely Fusarium graminearum MTCC-2093, F.

⁎ Corresponding author. E-mail address: [email protected] (S. Yadav). https://doi.org/10.1016/j.bioorg.2018.11.027 Received 23 March 2018; Received in revised form 8 November 2018; Accepted 17 November 2018 Available online 20 November 2018 0045-2068/ © 2018 Elsevier Inc. All rights reserved. D. Kumar et al. Bioorganic Chemistry 84 (2019) 24–31 pallidorosaum MTCC-2083, F. sabucinum MTCC-2085, F. moniliforme 2.5. α-L-Rhamnosidase purification MTCC-2088 and F. oxysporum MTCC-3075 were procured from the Microbial Type Culture Collection Center and Gene Bank, Institute of For the purification of the α-L-rhamnosidase from F. moniliforme Microbial Technology, Chandigarh (India) and were maintained on MTCC-2088, the fungal strain was grown in 20 sterilized 250 mL cul- agar strains of the media reported for them [19]. All these five fungal ture flasks each containing 25 mL liquid culture medium amended with strains were tested for the secretion of α-L-rhamnosidase in the liquid the 2.0% hesperidin and 4.0% sucrose. The α-L-rhamnosidase activity culture growth medium using the method reported in the literature reached the maximum value on 4th day from inoculation of the fungal [20]. Out of the above five fungal strains, only F. moniliforme MTCC- spores. On that day, the cultures of all the flasks were pooled, mycelia 2088 secreted α-L-rhamnosidase in the liquid culture growth medium. were removed by filtration through four layers of cheese cloth, andthe Hence further studies on the α-L-rhamnosidase of only F. moniliforme culture filtrate was centrifuged using Sigma refrigerated centrifuge MTCC-2088 were carried out. model 3K30 at 8000 rpm for 20 min at 4 °C to remove the particles. The centrifuged culture filtrate was concentrated by ultrafiltration using Amicon concentration cell model 8200 with PM10 membrane. The 2.3. α-L-Rhamnosidase secretion concentrated enzyme sample was dialyzed against 0.01 M of sodium phosphate buffer pH 7.0. The dialyzed enzyme was loaded on CM cel- The secretion of α-L-rhamnosidase by the fungal strain in the liquid lulose column equilibrated with the same buffer. The adsorbed enzyme culture growth medium was studied using the method reported by was washed with three times bed volume of the column and was eluted

Yadav et al. [20]. The liquid culture growth medium consisted of CaCl2 by applying the linear NaCl gradient of 0–1 M in the same buffer. The 1 g, MgSO4·7H2O 3 g, KH2PO4 20 g, N(CH2COONa)3 1.5 g, MnSO4 1 g, 3.5 mL fractions were collected and were analyzed for the activity of α- ZnSO4·7H2O 0.1 g, CuSO4·5H2O 0.1 g, FeSO4·7H2O 0.1 g, H3BO3 L-rhamnosidase [21] and for protein concentration using Lowry’s 10.0 mg, sucrose 40.0 g, ammonium tartrat8.0 g in 1L MilliQ Water. method [22]. The α-L-rhamnosidase active fractions were pooled and One mL of spore suspension (spore density 5 × 106 spore/mL) from the concentrated by putting the solution in a dialysis bag which was kept in agar slants of the fungal strain was inoculated aseptically into the solid powderedsucrose. The purified, concentrated enzyme sample was sterilized liquid culture growth medium (20 mL) kept in 100 mL culture stored in the refrigerator at 4 °C and was used whenever required. The flasks. The flasks were incubated in a B.O.D incubator at 25°Cunder enzyme did not loseits activity for two months under these conditions. stationary culture condition. Aliquots of one mL of the fungal growing cultures were withdrawn at the regular intervals of 24 h, filtered 2.6. SDS-polyacrylamide and native-polyacrylamide gel electrophoresis through millex syringe filters (0.22 mm) and analyzed for the presence of α-L-rhamnosidase activity by the method reported by Romero et al. The homogeneity of the enzyme preparation was checked by SDS- [21] as described below. The experiments were done in triplicates and PAGE analysis and the molecular mass was determined using the the average of enzyme unit/mL present in the growth medium was method reported by Weber and Osborn [23]. These parating gel was plotted against the fungus growth time in days. In order to enhance the 12% acrylamide in 0.375 M Tris–HCl buffer of pH 8.8 and stackinggel secretion of the enzyme in the above liquid growth medium by F. was 5% acrylamide in 0.5 M Tris–HCl buffer of pH 6.8. Proteins were moniliforme MTCC-2088, the liquid culture growth medium was visualized by silver staining. The molecular weight markers used were amended by adding 0.5 g of each of naringin, rutin, hesperidin, and L- phosphorylase B (97.4 kDa), bovine serum albumin (66.0 kDa), oval- rhamnose separately in four sets of experiments and the above experi- bumin (43.0 kDa), carbonic anhydrase (29.0) and soyabean trypsin ment was repeated. The maximum activity of α-L-rhamnosidase was inhibitor (20.1 kDa). The gel was run at the constant current of 20 mA observed in the medium amended with hesperidin. For the purification using Electro gel 50 equipment of Technosource, Mumbai, (India). The of the enzyme, the fungal culture was grown in the medium amended native polyacrylamide gel electrophoresis was done using the reagent with hesperidin. kitsupplied by Bangalore GENEI Pvt. Limited Bangalore (India). The resolving gel was 10% acrylamide in 0.5 M Tris–HCl buffer of pH 8.8 and the stacking gel was 5% acrylamide in 0.5 M Tris–HCl buffer of pH 2.4. α-L-Rhamnosidase assay 6.8.

The activity of α-L-rhamnosidase was assayed using p-nitrophenyl-α- 2.7. Steady-state kinetics of the enzyme L-rhamnopyranoside as the substrate and monitoring the liberation of p- nitrophenol spectrophotometrically at λ = 400 nm using molar ex- The values of Km and Vmax of the purified enzyme for the substrate p- −1 −1 tinction coefficient value of 21.44 mM cm by the reported method nitrophenyl-α-L-rhamnopyranoside were determined by measuring the [21]. 400 μL of 1 mM solution of p-nitrophenyl- α-L-rhamnopyranoside in steady state velocity of the enzyme catalyzed reaction at different a 0.5 M sodium phosphate buffer pH 10.5 was mixed with 500 μL ofthe concentrations of p-nitrophenyl-α-L-rhamnopyranoside (0.1–1.2 mM) same buffer in an Eppendoff tube. The tube was incubated inather- using the method reported by Romero et al. [21]. The Km and Vmax mostat maintained at 50 °C and was allowed to maintain thermal values were calculated by linear regression analysis of the data points of equilibrium. 100 μL of the enzyme sample was added to the Eppendoff the double reciprocal plot. The pH optimum of the purified enzyme was tube and mixed.100 μL reaction mixture was withdrawn immediately determined by using p-nitrophenyl-α-L-rhamnopyranoside as the sub- and added to 3.0 mL of 0.1 M NaOH solution. The aliquots of 100 μL of strate and measuring the steady state velocity of the enzyme catalyzed reaction mixture were withdrawn at regular intervals of 2 min and were reaction in solutions of varying pH in the range5.0–13.0. The buffers added to 3.0 mL of 0.1 M NaOH solutions kept in test tubes. The solu- used were 0.2 M sodium acetate/ acetic acid buffer solution (pH5–7), tions were allowed to stand for 10 min after which absorptions of so- and sodium phosphate buffer solution (pH8.0–13.0). The steady state lutions at λ = 400 nm were measured. All spectrophotometric mea- velocity was plotted against the pH of the reaction solutions and the pH surements were made with UV/Visible spectrophotometer Hitachi optimum was determined from the graph. The pH stability of the en- (Japan) model U-2000 which was connected to an electronic tem- zyme was determined by incubating theenzyme in the buffers of dif- perature control unit. The least count of the absorbance measurement ferent pHs for 24 h at 25 °C. The remaining activities were assayed and was 0.001 absorbance unit. One enzyme unit was the amount of the plotted against pH to which the enzyme was exposed for 24 h. The enzyme which liberated 1 μmol of p-nitrophenol/min at 50 °C tem- temperature optimum was determined by measuring the steady state- perature under the assay conditions specified above. velocity of the enzyme catalyzed reaction in solutions of varying temperatures (30–70 °C) using p-nitrophenyl-α-L-rhamnopyranoside as the

25 D. Kumar et al. Bioorganic Chemistry 84 (2019) 24–31 substrate. The steadystate velocity of the enzyme catalyzed reaction was plotted against the temperature of the reaction solution and the temperature optimum was determined from the graph. The thermal stability of the enzyme was determined by incubating the aliquots of theenzyme at different temperatures (viz. 10, 20, 30, 35, 40, 45, 50, 55, 60, 65 and 70 °C) and assaying the remaining activity at the intervals of 10 min for 2 h. The remaining activity was plotted against the time for which the enzymes were exposed at that temperature.

2.8. Studies on the enzymatic hydrolysis of naringin, hesperidin and rutin

This experiment was performed in 1.0 mL solution of 1.0 mM naringin in 0.5 M sodium phosphate buffer pH 10.5 at 30 °C. 25 μL purified enzyme stock (0.30 U/mL) was added. The reaction solution was left overnight and the release of prunin was detected by thin layer chromatography using silica gel on glass plates and using butanol:acetic acid:water (40:11:29) (v/v) as mobile phase. The detection was made Fig. 1. Secretion of α-L-rhamnosidase in the culture medium of Fusarium mon- using the iodine chamber. Similar experiments were performed with iliforme MTCC-2088. Hesperidin (■), naringin (▴), control (●) bagasse (△) and rutin and hesperidin. Preparative TLC in silica gel on glass plates were corncob (□). used for purifying the enzymatic hydrolysis products of naringin, rutin and hesperidin for HPLC-Mass spectrometric analysis. The HPLC-Mass 6.0 U/mg and 13.0 U/mg, respectively. The value of specific activity of Spectrometric analysis was done at Sophisticated Analysis Instrument this enzyme is comparable to the specific activity of α-L-rhamnosidases Centre, CDRI, Lucknow, U.P. (India). The release of L-rhamnose was reported from A. awamori MTCC-2879 [26] and P. citrinum MTCC-3565 also detected by the thin layerchromatography using silica gel on glass [27] both of which have specific activity 27 U/mg. However, the spe- plate and chloroform: methanol (70:30) (v/v) as the mobile phase. The cific activity of this enzyme is less than specific activity of α-L-rham- L-rhamnose released as the results of enzymatic hydrolysis of naringin, nosidase reported from P. citrinum MTCC-8897 [28] 42.7 U/mg. The rutin and hesperidin also was purified by preparative TLC and was purification procedure is relatively simpler than the procedures re- analyzed by HPLC-Mass Spectrophotometric studies. ported for the purifications of α-L-rhamnosidases from other sources [2]. 2.9. Studies on the enzymatic release of L-rhamnose from wine sample The results of SDS-PAGE and native PAGE analysis are shown in Fig. 3A and B, respectively. The presence of a single protein band in In 1.0 mL of the wine sample, 0.30 U of the purified enzyme from F. lane 2 of Fig. 3A in which the purified enzyme was applied showed that moniliforme MTCC-2088 was added. The reaction solution was left the enzyme was pure. The purity of the enzyme was further confirmed overnight, and the release of L-rhamnose was detected by thin-layer by the presence of a single protein band in lane 1 of the native-PAGE chromatography using silica gel on glass plates. The mobile phase used analysis result shown in Fig. 3B in which the purified enzyme was ap- was chloroform: methanol mixture 70:30 (v/v). The detection was plied in lane 1 and the reference protein, bovin serum albumin, which made using the iodine chamber. Again the identity of L-rhamnose re- was applied in lane 2. The molecular mass of the purified α-L-rham- leased by the enzymatic hydrolysis was confirmed by HPLC-Mass nosidase calculated from the SDS-PAGE analysis was 36.0 kDa. The Spectrophotometric studies of the product which was purified by pre- molecular mass of the purified Lα- -rhamnosidases reported in the lit- parative TLC. erature [2] are in the range 41.0–240 kDa. Thus the purified α-L- rhamnosidase is one of the α-L-rhamnosidases having lower molecular 3. Results and discussion mass.

3.1. Secretion and purification of theL α- -rhamnosidase 3.2. Kinetics characteristics of the enzyme In order to enhance the secretion of α-L-rhamnosidase in the liquid culture growth medium, the effects of presence of naringin, rutin, he- The kinetic characteristics of the purified enzyme were studied speridin and L-rhamnose in the growth medium on the secretion of α-L- using p-nitrophenyl-α-L-rhamnopyranoside as the substrate. The en- rhamnosidase were studied. The results are shown in Fig. 1. All the zyme followed the Michaelis-Menten kinetics (data not shown here) the three substrate, naringin, rutin, hesperidin and one product L-rhamnose, values of Km and kcat calculated from the double reciprocal plots were −1 enhance the secretion of α-L-rhamnosidase in the growth medium. 0.5 mM and 15.6 s at 50 °C in 0.2 M sodium phosphate buffer pH 10.5 However, the enhancement of α-L-rhamnosidase secretion in the growth using p-nitrophenyl-α-L-rhamnopyranoside as the substrate. The kcat/Km medium containing hesperidin was maximum. Since the structure and value of the purified enzyme under the above condition is 4 −1 −1 4 regulation of α-L-rhamnosidasegenes is not fully understood [24], the 3.12 × 10 M s which is lower by a factor 10 in comparison to a reason for enhancement of α-L-rhamnosidase secretion in the presence perfectly evolved enzyme [29]. Thus there is scope for improving the of the above substrates and the products cannot be discussed further. catalytic efficiency of this enzyme using the molecular biology techni- The purification procedure of α-L-rhamnosidase from the culture filtrate ques of directed evolution [30,31]. of F. moniliforme MTCC-2088is summarized in Table 1 and the elution profile of the enzyme from the cation exchanger carboxymethyl cellu- 3.3. pH and temperature optima and stabilities lose is shown in Fig. 2. The enzyme bound to CM cellulose in 10 mM sodium phosphate buffer pH7.0 and was eluted by the linear gradient of The results of studies on the variation in the activity of the enzyme NaCl in the range 400–660 mM. About6 fold purification with 19% with the variation in the pH of the reaction solution are shown in yield was achieved. The specific activity of the purified enzyme using p- Fig. 4A. The pH optimum of the enzyme was 10.5 at pH 50 °C. Thus it is nitrophenylα-L-rhamnopyranoside is 26 U/mg is greater than the spe- one of the α-L-rhamnosidase which has pH optimum in very alkaline cific activity of α-L-rhamnosidases reported from A. clavato-nanicus range. There are a few reports on α-L-rhamnosidases having pH optima MTCC-9611 [20] and P. corylopholum MTCC-2011 [25], the value being in alkaline range i.e. α-L-rhamnosidase from Clostridium stercorarium

26 D. Kumar et al. Bioorganic Chemistry 84 (2019) 24–31

Table 1 Purification chart α-L-rhamnosidase from Fusarium moniliforme MTCC-2088.

S. no. Steps Total volume Activity U/mL Protein mg/ Total activity Total protein Specific activity U/ Protein fold % Yield mL mL U mg mg

1 Culture filtrate of the fungal strain 600 0.095 0.023 57 13.8 4.13 1 100 2 After Amicon concentration 10 4.82 0.81 48.2 8.1 5.95 1.14 84 3 After Dialysis 12 4.01 0.60 48.12 7.2 6.68 1.16 84 4 After CM cellulose column 10.5 1.04 0.04 10.92 0.42 26 6.3 19 concentration

that pH and temperature optima are not always independent of each other as is assumed in traditional enzymology. To check this point, the pH optima of the purified enzyme have been determined at two addi-

Pr ot e tional temperature 40 °C and 60 °C using naringin as the substrate [2]. The results are shown as insert (B) in Fig. 4(a). The pH optima of the /min) ( A in ( enzyme at 40 °C is 9.5 pH unit were as at 60 °C it is 10.5 pH unit. The pH 400

A stability of the purified enzyme was also studied for the point of view Δ 750

) determination of pH at which the enzyme could be preserved. The results are shown in Fig. 4B in which residual activity of the enzyme

( Ac tivity exposed for 24 h at a particular pH has been plotted against the pH at which the enzyme has been exposed for 24 h. The enzyme is most stable between pH 9.0–10.0. Thus the most suitable pH for preserving the enzyme is between 9.0 and 10.0. The data on the pH stabilities of α-L- rhamnosidases are not available in literature [2] for comparison. Fraction Number The results of the studies on the variation of the activity of the enzyme with variation of temperature of the reaction solution are Fig. 2. Elution profile of α-L-rhamnosidase from CM-cellulose column. Protein shown in Fig. 5A. It can be seen that the temperature optimum of the concentration (A )(■); enzyme activity (A /min) (●); NaCl gradient 750 400 enzyme is 50 °C which is in the range of temperature optima (40–80 °C) ( − − − −). reported in the literature [2] for other α-L-rhamnosidases. The thermal stability of the enzyme has been studied with the objective of de- (pH7.5), α-L-rhamnosidase from PRI-1686 RhmA (pH7.9), α-L-rhamno- termining the suitable temperature at which the enzyme could be sidase of Pseudomonas paucimobilis FP 2001 (pH 7.8) [2] and α-L- preserved. The results are also shown in Fig. 5B, in which the remaining rhamnosidases from A. terreus (pH 8.0) [12], but all these α-L-rhamno- activity of the enzyme exposed for 1 h at a particular temperature has sidases have pH optima 8.0 or less than. It is also essential to mention

66.0 kDa 97.4 kDa

66.0 kDa

44.0 kDa

Purified enzyme 36.0 kDa 29.0 kDa

Purified enzyme 36.0 kDa 20.0 kDa

1 2 1 2 (B) (A)

Fig. 3. Results of gel electrophoresis (A) SDS-PAGE and (B) Native-PAGE: (A) Lane 1 molecular weight markers and lane 2 purified enzyme. (B) Lane 1 purified enzyme and lane 2 bovine serum albumin.

27 D. Kumar et al. Bioorganic Chemistry 84 (2019) 24–31

Fig. 4. Effect of pH on the activity of the en- 5 zyme. (A) pH optimum at 50 °C (●). (B) pH op- 4 timum at 40 °C and 60 °C (C) pH stability of en- (B) 3 zyme (○). (A) The assay solution 1.0 mL contained 0.4 mM substrate, 1.50 μg of the pure 2 (A) enzyme in sod. Phosphate buffer of varying in 1 the range 5–13 at 50 °C. (B) The assay solution of 0 varring pH in the range 7–13 left overnight at Activity ( µ mole / min) / ( mole µ Activity 8 10 12 25 °C and analyzed for the activity after 24 h. pH

(C)

derhamnosylated product of naringin neither coincides with the Rf value of naringin nor with the value of aglycon naringenin. It has been inferred that the enzymatically derhamnosylated product of naringin is prunin. Further confirmation of the identity of the product prunin was done by purifying it by preparative TLC and analyzing by HPLC-Mass spectrometry (Fig. 6B). The hydrolysis products of rutin and hesperidin were also analyzed in the same way as the enzymatic hydrolytic product of naringin. The results are given in Fig. 6C and D respectively. Rutin is converted to isoquercitrin and hesperidin is converted to hesperetin glucoside.

3.5. Enzymatic release of L-rhamnose from the wine sample

The enzymatically released L-rhamnose from the wine sample was analyzed in the same way as L-rhamnose released in the enzymatic hydrolysis of naringin, rutin and hesperidin and the result is given in Fig. 7(A and B). Fig. 7B shows the results of TLC experiment on wine sample (lane 1), wine sample treated with enzyme (lane 2) and stan- Fig. 5. Effect of temperature on the activity of enzyme. (A) Temperature dard sample of L-rhamnose. There is no free L-rhamnose in wine sample Optima (■). The assay solution 1.0 mL continued 0.4 mM substrate, 1.50 μg of but when wine sample is treated with enzyme L-rhamnose is released. pure enzyme in 0.5 M sodium phosphate buffer pH 10.5 at varying temperature The result of HPLC-Mass analysis of the purified L-rhamnose by pre- (30–70 °C). (B) Thermal stability (●). The assay solution at varying temperature parative TLC is shown in fig.B. The presence of molecular ion peakat (10–70 °C) left for one hour and analyzed for the remaining activity. m/z = 164.2 confirms the identity of L-rhamnose. The derhamnosylation of wine ingredients is related to aroma en- been plotted against the temperature at which the enzyme was exposed hancement of wine. The volatile components such as linalool, geraniol, for 1 h. The results show that the enzyme starts loosing activity even at nerol, citronellol and terpinol are responsible for the aroma of wine 10 °C but it is more pronounced above 30 °C. Thus a suitable tem- (2,17,18). Most of these components are present in grape skin as perature for preserving the enzyme is below 10 °C. The energy of acti- odourlessdiglycosides of terpenes, viz. α-L-arabinofuranosyl-β-D-gluco- vation for thermal denaturation of the enzyme was also determined and pyranosidases and α-L-rhamnopyranosyl-β-D-glucopyranosides which on it was 22.09 kJ/mol. On the basis of the above studies, the purified two sequential hydrolysis release volatile terpinol. The immobilized α-L- enzyme was preserved in a solution of pH10 at 4 °C in the refrigerator. rhamnosidase along with β-glucosidase and α-arabinosidase has been used for the aroma enhancement of wine [32]. Thus the purified α-L- rhamnosidase has also a potential in the aroma enhancement of wine. 3.4. Derhamnosylation of naringin, rutin and hesperidin

The results of studies on the enzymatic de-rhamnosylation of nar- 4. Conclusions ingin, rutin and hesperidin are shown in Fig. 6A. The spots in lane 1 and lane 2 are due to the standard sample of naringin and its aglycon This communication reports the purification of an α-L-rhamnosidase naringenin, respectively, whereas the spot in lane 3 is due to the en- from the culture filtrate of a novel fungal strain F. moniliforme MTCC- zymatic de-rhamnosylated product of naringin. The Rf value of the 2088, using a simpler procedure. The enzyme has pH optima of 10.5

28 D. Kumar et al. Bioorganic Chemistry 84 (2019) 24–31

(A)

1 2 3 4 5 6 7 8 9

(B)

(caption on next page)

29 D. Kumar et al. Bioorganic Chemistry 84 (2019) 24–31

Fig. 6. Identification of de-rhamnosylated products of naringin, rutin and hesperidin: (A). TLC Chromatogram of naringin, rutin, hesperidin, their derhamnosylated product and their aglycons. Naringin (lane 1), naringenin (lane 2), reaction product of naringin (lane 3), pure rutin (lane 4), reaction product of rutin (lane 5) and pure quercetin (lane 6), reaction product of hesperidin (lane 7), hesperidin (lane 8) and hesperetin (lane 9). (B) HPLC-Mass analysis result of the de-rhamnosylated product of naringin (prunin). (C) HPLC-Mass analysis result of the de-rhamnosylated product of rutin (isoquercetrin). (D) HPLC-Mass analysis result of the de- rhamnosylated product of hesperidin (hesperetin-glucoside).

Fig. 6. (continued)

30 D. Kumar et al. Bioorganic Chemistry 84 (2019) 24–31

Biochem. 45 (2010) 1226–1235. [3] H.Y. Chang, Y.B. Lee, H.A. Bae, J.Y. Huh, S.H. Nam, H.S. Sohn, H.J. Lee, S.B. Lee, Purification and characterization of Aspergillus sojae naringinase: the production of prunin exhibiting markedly enhanced solubility with in vitro inhibition of HMG- CoA reductase, Food Chem. 124 (1) (2011) 234–241. [4] Ni H. Xia, A.F. Cai, H.N. Chen, F. You, Y.Z. Lu, Purification and characterization of Aspergillus niger α-L-rhamnosidase for the biotransformation of naringin to prunin, African J. Microbiol. Res. 6 (24) (2012) 5276–5284. [5] Y.S. Lee, J.Y. Huh, S.H. Nam, S.K. Moon, S.B. Lee, Enzymatic bioconversion of citrus hesperidin by Aspergillus sojae naringinase: enhanced solubility of hesperetin-7-O- glucoside with in vitro inhibition of human intestinal maltase, HMG-CoA reductase, and growth of Helicobacter pylori, Food Chem. 135 (4) (2012) 2253–2259. [6] G. Celiz, J. Rodriguze, F. Soria, M. Daz, Synthesis of hesperetin 7-O-glucoside from flavonoids extracted from citrus waste using both free and immobilized α-L- rhamnosidases, Biocatal. Agric. Biotechnol. 4 (3) (2015) 335–341. [7] K. Valentova, J. Vrba, M. Bancirova, J. Ulrichova, V. Kren, Isoquercitrin: pharma- cology, toxicology, and metabolism, Food Chem. Toxicol. 68 (2014) 267–282. [8] J. Wang, A. Gong, C. Yang, B. Feng, Si, Shi, Xin-Yi, Han, Bei-Bei, Wu, Xiang-Yang 1 2 3 and Wu, Fu-An. Biosyn. Biocat., 2015 (/5:8682/doi:10.1038/orep 08682,1-8). [9] J. Wang, A. Gong, S. Gu, H. Cui, X. Wu, Ultrafast synthesis of isoquercitrin by (A) enzymatic hydrolysis of rutin in a continuous-flow micro reactor, J. Serb. Chem. Soc. 80 (7) (2015) 853–866. [10] K. Valentova, P. Sina, Z. Rybkova, J. Krizan, K. Malachova, V. Kren, Antimutagenic and immunomodulatory properties of quercetinglycosides, J. Sci. Food Agri. 96 (5) (2016) 1492–1499. [11] E. Vavríkova, F. Langschwager, L. Jezova-Kalachova, A. Krenková, B. Mikulová, M. Kuzma, V. Křen, K. Valentová, Isoquercitrin esters with mono- or dicarboxylic acids: enzymatic preparation and properties, Int. J. Mol. Sci. 17 (6) (2016) 899, https://doi.org/10.3390/ijms17060899. [12] T. Makino, R. Shimizu, M. Kanemaru, Y. Suzuki, M. Moriwaki, H. Mizakami, Enzymatic modified isoquercitrin, α-oligoglycosylquercetin3-O-glucoside, is ab- sorbed more easily than other quercetin glycosides or aglycon after oral adminis- tration in rats, Biol. Pharma. Bull. 32 (2009) 2034–2040. [13] N. Masuka, I. Kuba, Characterization of the antioxidant activity of flavonoids, Food Chem. 131 (2012) 541–545. [14] L. Weignerova, P. Marhol, D. Gerstorferova, V. Kren, Preparatory production of quercetin-3-β-D-glucopyranoside using alkali-tolerant thermostable α-L-rhamnosidase from Aspergillusterreus, Biores. Technol. 115 (2012) 222–227. [15] D. Gerstorferova, B. Fliedrova, P. Halada, P. Marhol, V. Kren, L. Weignerova, Recombinant α-L-rhamnosidase from Aspergillusterreus in selective trimming of rutin, Process Biochem. 47 (5) (2012) 828–835. [16] M. Rebros, A. Pilnikova, D. Simcíkova, M. Rosenberg, Recombinant α-L-rhamnosidase of Aspergillusterreus immobilization in polyvinylalcohol hydrogel and its application in rutin derhamnosylation, J. Biocat. Biotrans. 31 (2013) 329–334. [17] M. Orejas, E. Ibanez, D. Ramon, The filamentus fungus Aspergillus nidulans produces (B) an α-L-rhamnosidase of potential oenological interest, Lett. Appl. Microbiol. 28 (1999) 383–388. [18] P. Manzanares, M. Orejas, J.V. Gil, L.H. Graaff, J. Visser, D. Ramon, Construction of Fig. 7. Results of the release of L-rhamnose from enzyme treated wine. (A) TLC a genetically modified wine yeast strain expressing the Aspergillus aculeatus rhaA Chromatogram of wine (lane 1), wine treated with pure enzyme (lane 2) and gene, encoding anα-L-rhamnosidase of enological interest, Appl. Environ. standard sample of L-rhamnose. (B) HPLC-Mass analysis result of the drham- Microbiol. 69 (2003) 7558–7562. nosylated product L-rhamnose. [19] Catalogue of Strain, Published by Microbial Type Culture Collection Center and Gene Bank Institute of Microbial Technology, Chandigarh, India, 2000. [20] Y. Vinita, Y. Saroj, Y. Sarita, K.D.S. Yadav, α-L-rhamnosidase from which is suitable for derhamnosylation of naringin, rutin, and hesper- Aspergillusclavato-nanicus MTCC-9611 active at alkaline pH, Appl. Biochem. Microbiol. 48 (3) (2012) 295–301. idin. [21] C. Romero, A. Manjon, J. Bastida, J.L. Iborra, A method for assaying rhamnosidase activity of naringinase, Anal. Biochem. 149 (1985) 566–571. [22] O.H. Lowry, N.J. Rosebrough, A.L. Farrand, R.J. Randall, Protein measurement with Disclosure statement the folin-phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [23] K. Weber, M. Osborn, The reliability of molecular weight determination by dodecyl No potential conflict of interest was reported by the authors. sulfate-polyacrylamide gel electrophoresis, J. Biol. Chem. 244 (1969) 4406–4412. [24] Tamayo-Ramos&Orejas, Enhanced glycosyl hydrolase production in Aspergillusnidulans using transcription factor engineering approaches, Biotechnol. Acknowledgements Biofuels 7 (2014) 103–112. [25] S. Yadav, S. Yadava, K.D.S. Yadav, Purification and characterization of α-L-rhamnosidase from Penicilliumcorylopholum MTCC-2011, Process Biochem. 48 (9) Dr. SaritaYadav is thankful to UGC, New Delhi, for the award of a (2013) 1348–1354. Post Doctoral Fellowship for Women no. PDFWM-2014-15-OB-UTT- [26] S. Yadav, R.S.S. Yadava, K.D.S. Yadav, Purification and characterization of α-L- rhamnosidase from Aspergillusawamori MTCC 2879, I. J. Food Sci. Technol. 48 23606. Dhirendra Kumar acknowledges the financial support of UGC, (2013) 927–933. New Delhi in the form of a Rajeev Gandhi National Fellowship no. F1- [27] S. Yadav, V. Yadav, S. Yadava, K.D.S. Yadav, Purification, characterization and 17.1/2016-17/RGNF-2015-17-SC-UTT-2100/(SA-III). Prof. K.D.S. application of α-L-rhamnosidase from Penicilliumcitrinum MTCC-3565, I. J. Food Sci. Technol. 47 (2012) 1404–1410. Yadav and Prof. SudhaYadava acknowledges the financial support of [28] S. Yadav, V. Yadav, S. Yadava, K.D.S. Yadav, Purification, characterization and UGC, New Delhi in the form of a major research project file no.37/134/ application of α-L-rhamnosidase from Penicilliumcitrinum MTCC-8897, I. J. Food Sci. Technol. 47 (2012) 290–298. 2009(SR). [29] A. Fersht, Structure and Mechanism in Protein Science: A Guide to Enzymecatalysis and Protein Folding, WH Freeman and Company, New York, 1999. References [30] F.H. Arnol, G. Georgiou, Directed enzyme evolution: library creation. Methods and protocols, Humana Press, Totowa, New Jersey, 2003. [31] F.H. Arnol, G. Georgiou, Directed Enzyme Evolution: Screening and Selection [1] P. Manazanres, S. Vales, D. Ramon, M. Orejas, α-L-Rhamnosidase: old and new Methods, Humana Press, Totowa, New Jersey, 2003. insights, in: J. Polaina, A.P. MacCabe (Eds.), Industrial Enzymes, Springer Verlag, [32] C. Caldini, F. Bonomi, P.G. Pifferi, G. Lanzarini, Y.M. Galante, Kinetic andim- Berlin-Heidelberg, 2007, pp. 117–140. mobilization studies on the fungal glycosidases for the aroma enhancement in wine, [2] V. Yadav, P.K. Yadav, S. Yadav, K.D.S. Yadav, α-L-Rhamnosidase: a review, Process Enz. Microb. Technol. 16 (1994) 286–291.